Breast cancer (BC) continues to be the most common cancer in women and is responsible for almost 40,000 deaths per year. According to the VA Center for Women Veterans, women are the fastest growing subgroup of U.S. veterans and this number is expected to grow in the next 10 years. Therefore, as the population ages, VA health care will be in high demand by women veterans. This is because some of the same risk factors that are associated with poor prognosis in the general population are also found in the VA population. The VA also treats a moderately high volume of male BC and, unlike the declining mortality for female BC, death rates from male BC have not decreased. Compared with the general population, a higher proportion of the male and female BC patients in the VA system are African-American, relative to the private sector, and this population exhibits higher mortality and morbidity rates. Therefore, identifying effective therapeutic targets to treat these diverse groups of veterans is highly relevant to the VA patient care mission. The previous VA Merit funded project addressed questions about how breast cancers acquire hormone independence and become metastatic or spread throughout the body. We suspected that this type of tumor progression was controlled by a BC protein (RUNX2) that regulates expression of genes that promote growth and metastasis. We also proposed to discover compounds that would inhibit the ability of this factor to increase BC progression. We found that the RUNX2 factor promotes BC progression to a more aggressive, metastatic form by increasing glucose metabolism, inhibiting mitochondrial respiration, and reducing epithelial- specific gene expression, or differentiation. We also used novel drug screening methods and activity assays developed in the laboratory to characterize several compounds related to vitamin D that inhibited the RUNX2 factor and BC growth. Given these observations, in this VA Merit renewal application we propose the hypothesis that the RUNX2 factor can alter BC metabolism and inhibit cell differentiation while promoting tumor progression. We further propose that targeted inhibition of this factor using novel vitamin D derivatives will restore normal cell metabolism and differentiation, prevent further tumor progression, and inhibit BC growth. The goal of this proposal, therefore, is to perform a comprehensive but focused analysis of the relationship between RUNX2-mediated breast cancer growth and differentiation. RUNX2 may promote a metabolic progression favoring glycolysis and suppressing mitochondrial respiration. Several specific BC cell models and novel compounds that promote BC cell differentiation will be used to define the mechanisms regulating breast cancer progression and metastasis. The goals are to (1) define the RUNX2-regulated relationship between BC cell differentiation, metabolism, and progression, (2) define how Vitamin D3 prohormone regulates RUNX2 DNA binding and BC differentiation through its regulation of cell metabolism, and (3) determine the mechanisms regulating RUNX2 DNA binding and BC differentiation by oxidized variants of Vitamin D3. Many patients within the Veterans Administration health care system also suffer from lung, colorectal, and head/neck cancers, which are prevalent in elderly veterans who are smokers. Our study may provide insight into understanding the molecular pathways regulating these malignancies. Because the hypotheses we are testing are also relevant to how RUNX2 regulates vascular disease, therapeutic targeting strategies discovered in this study may have applicability and clinical relevance for other pathologies prevalent in the veteran population that are exacerbated by aging and smoking, such as cardiovascular disease. !